Taste has been studied in animals and humans at behavioral, neuroanatomical, physiological and cellular levels. Comprehensive descriptions at the molecular level have not yet been reported. This proposal is aimed at remedying this deficit. Glutamate (as monosodium glutamate, MSG) is a potent taste stimulus for humans and other mammals and is presumed to bind to specific receptor proteins at the exposed tips of chemosensory cells in taste buds. Project #1 of this Program Project will serve as the molecular biological foundation of a multi-disciplinary study of the molecular, cellular and functional definition of glutamate receptors significant in the transduction of glutamate taste. Using the brain glutamate receptors as a starting point, we propose to search for glutamate receptors that are expressed in taste buds and clone full-length cDNAs representing these. This will be achieved by employing degenerate PCR primers to amplify sequences representing glutamate receptors closely or distantly related to those in the brain. Cloned PCR products will be classified, using RNase protection and Northern blots, to identify sequences uniquely expressed in taste buds rather than in surrounding epithelium. Appropriate partial cDNA clones will then serve as probes for isolating full-length cDNA clones. We will analyze the predicted primary structure of the taste cell glutamate receptors to verify that they conform to the overall transmembrane topology for other, known glutamate receptors. We will also search for primary sequence motifs which would relate to functional characteristics of the encoded receptors (e.g. permeability of the ion channel to Ca++, binding site for ribonucleotide monophosphates and glutamate-binding site). We will assist Project #2 by providing Dr. Roper with probes for in situ hybridization and with subcloned cDNAs from which to generate probes. We will also assist Project #2 in the design of synthetic peptides for immunization. The studies will lead to the cellular localization of mRNA and protein for taste-specific glutamate receptors. We will assist Project #3 by producing in vitro transcribed full-length mRNA for injection into Xenopus oocytes and by expressing the full-length cDNAs in mammalian cell lines. These will be used to ascertain the functional properties of the cloned glutamate receptors. The proposed studies will thus comprise a coordinated search for novel glutamate receptors significant for the biology of taste.